Project Summary/Abstract Molecular imaging through PET/CT is an indispensable tool for the detection, characterization and monitoring of tumors and therapies. The most widely used PET/CT molecular imaging agent is the glucose analog 18F-fluorodeoxyglucose (18F-FDG). Some tumors are glycolytic (the Warburg Effect) and therefore accumulate 18F-FDG, however, there is non-specific uptake in several tissues and not all tumors are glycolytic. Thus, targeting a more ubiquitous tumor biomarker would provide an advantage over existing PET/CT agents. The goal of this project is to develop novel Scanning Unnatural Protease Resistant (SUPR) peptide- based radiotracers to detect the expression of tumor Mucin 1 (MUC1) by PET/CT. SUPR peptides are developed with mRNA display, an in vitro selection technology where translated peptides are linked covalently to their encoding mRNAs. Compared to antibodies, peptide agents have increased tumor penetration, retention and faster systemic clearance. This increases tumor signal, decreases background and allows same-day imaging. MUC1 is a heavily O-glycosylated transmembrane protein normally expressed on the apical surface of secretory and glandular epithelium. MUC1 is overexpressed in >90% of carcinomas, and more strongly expressed in metastatic lesions. The MUC1 ectodomain contains 25-125 tandem repeats (TR) of 20 amino acids. In normal cells, the TR is decorated with extended, branched, core-2 glycosylation patterns, whereas cancer cells decorate with shorter, unbranched core-1 chains (absent in normal tissues). Thus, molecular imaging of tumor glycosylated MUC1 would provide near-universal detection of tumors with low non-specific background. We will synthesize MUC1 TR with solid phase synthesis and perform in vitro glycosylation with recombinant glycosyltransferases. This will produce a chemically-defined T antigen expressing MUC1 TR (T-TR peptide) for selecting SUPR peptides. We will screen a trillion member mRNA display library for binding to T-TR peptide and protease resistance to select for a highly stable (serum half-life of hours), high affinity (nM) SUPR peptides. Top candidates will be tested for in vitro stability, affinity to T-TR peptide and conjugated to Cy5 for flow cytometry. Affinity of SUPR peptides to core-1 expressing cell line T47D, CRISPR/Cas T47D-MUC1 knockout, sialyl-T-antigen decorated CHO-K1 and core-2 expressing cell line MCF7 will verify MUC1- and glycosylation-specificity. Affinity and specificity will be compared to core-1-specific MUC1 antibody, SM3. Then, we will test in vivo tumor uptake, non-selective retention, and clearance mechanisms of Cy5-SUPR in subcutaneous xenograft models. Finally, optimal SUPR peptides will be labeled with 18F and tested in PET/CT. These studies will produce high-affinity, high-specificity, and near-universal radiotracers for PET/CT. Non-invasive detection of MUC1 may allow more accurate, cost-effective patient stratification, improving clinical decisions and patient outcome.